1. Field of the Invention
This invention relates to the field of specific binding assays, particularly to overcoming non-specific protein interference in agglutination assays of non-protein ligands. Agglutination assays are highly sensitive and used to determine a wide variety of substances. These assays have been embodied in commercially available test kits such as those used to detect triiodo-L-thyronine (T.sub.3) and/or thyroxine (T.sub.4).
2. Brief Description of the Prior Art
The development of specific binding assay techniques has provided extremely useful analytical methods for determining various organic substances of diagnostic, medical, environmental and industrial importance which appear in liquid mediums at very low concentrations. Specific binding assays are based on the specific interaction between a ligand, i.e., a bindable analyte under determination, and a binding partner therefor, i.e., receptor. Where one of the ligand and it binding partner is an antibody and the other is a corresponding hapten or antigen, the assay is known as an immunoassay.
These specific binding assays have been provided in a variety of solid state formats including analytical elements or test strips, coated tubes, particle-associated reagents and others. Agglutination assays are among the most widely used solid state specific binding assays, usually as immunoassays. They may be classified as direct, indirect (passive) or inhibition type agglutination assays. In a direct agglutination assay, particles having surface components which are one member of a specific binding pair (e.g., a receptor), are reacted with a sample to be assayed for the other member of the specific binding pair (e.g., ligand). In the indirect (passive) agglutination format, one member of a specific binding pair (e.g., receptor) is bound to a solid substrate particle, and this particle-bound member is reacted with a sample to be assayed for the other member of the pair (e.g., ligand). In inhibition-type agglutination assays, a sample to be tested for one binding pair member is first reacted with a solution containing the other member of the binding pair and this prereacted solution is then reacted with particles which contain (direct) or are bound with (indirect) the binding pair member suspected of being in the sample. Agglutination assays have been summarized in the literature. See, for example, Bellanti, Immunology, W. B. Saunders Co., Philadelphia (1971), pgs. 139 et seq; and Fudenberg, et al, Basic & Clinical Immunology, Lange Medical Publications, Los Altos, CA. (1976), pp. 308 et seq. Also, Sawai et al, U.S. Pat. Nos. 4,118,192 and 4,208,185 relate to agglutination assays. Earlier references which are likewise relevant are Singer et al, J. Colloid and Interface Science, 45:608-614 (1973) and Faure et al, Protides of the Biological Fluids, Proceedings of the Colloquium, 20:589-593 (1972). A number of agglutination assay test kits for specific analytes or ligands are commercially available and have also been described in the literature. See, for example, Rose, et al (Eds.), Manual of Clinical Immunology, American Society for Microbiology, Washington, D.C. (1978).
When assaying complex liquids, such as human serum, many non-specific proteins, such as lipoproteins and autoantibodies, inhibit the agglutination reaction. Therefore, these proteins must be destroyed to obtain accurate measurement of ligand concentrations. The prior art has thus far required a pretreatment procedure separate from the assay. For Example, Kobayashi, et al, Steroids, 34:829-834 (1979), discloses a direct fluorescence polarization immunoassay of serum cortisol in which non-specific serum protein binding of fluorescent-labeled hapten (cortisol) was eliminated by sodium dodecyl sulfonate (SDS). The SDS was not removed prior to performing the assay.
Non-specific protein interference in assays for nonprotein ligands can be overcome by first digesting the proteins using a proteolytic enzyme such as pepsin. The enzyme is then inactivated or destroyed prior to the assay. For example, Collet-Cassart, et al Clin.Chem., 27:1205-09 (1981) disclose a particle-counting immunoassay (PACIA) for digoxin in samples which were predigested with pepsin. The digestion was stopped by adding tris(hydroxymethyl)methylamine which inactivates the pepsin. See also Chau et al, J. Clin. Endocrinol. Metab., 42:189-192 (1976).
Agglutination assays so far available, including those for the determination of T.sub.3 and T.sub.4, have suffered from non-specific protein interference. Invariably, it has been necessary to perform preliminary procedures to overcome this source of interference. Thus, despite the efforts reflected in the above references, no one has met the problem of providing a specific binding agglutination assay which avoids the effects of this interference without the need for pretreatment.